In 1966 and early 1967, two teams of investigators, working independently in the United States and in Sweden, were able to define the exact nature of the skin sensitizing antibody. The antibody proteins were found to belong to a new class of immunoglobulins which were officially designated immunoglobulin E in 1968. Within months after the discovery of the nature of IgE, new radioimmunoassays were developed for its detection.
Radioimmunoassays have earned a definitive place in clinical laboratory practice and are currently used for the detection of vitamins, enzyme systems, viruses, drugs, and at least 20 polypetide hormones. The assays derived their fundamental value from two separate properties--their sensitivity and their specificity. Any specific substance against which a specific antibody can be produced can be measured at concentrations to a billionth of a milligram.
There are two commercially available radioimmunoassays which are used in the diagnosis and management of allergy. They are the PRIST test and the RAST test.
The PRIST test measures total IgE. A paper disc to which an anti-IgE has been bound is incubated with a drop of the patient's serum. This disc binds all of the IgE in the sample. The disc is then washed to remove extraneous materials and radioactively labelled anti-IgE is added for a second incubation. During this time, the labeled anti-IgE reacts with IgE molecules previously bound to the disc and after a final washing step, the amount of radioactivity bound to the disc is measured in a gammacounter. The amount of radioactivity binding the test serum is then compared to the binding obtained by serial dilutions of a PRIST reference standard known to contain exactly 100 units of IgE. The use of a suitable reference standard is a basic requirement of all radioimmunoassay determinations because there can be unexpected variables based on changes in incubation time, changes in room temperature, and decay in the amount of radioactivity bound to the anti-IgE used in the second stage.
The RAST test is a measurement of a specific allergen. The allergen of interest, such as short ragweed, is bound to the disc and reacts only with the short ragweed IgE in the sample. After the initial incubation, non-specific IgE antibody and other proteins are removed by washing. Radioactively-labeled anti-IgE is then added and allowed to incubate overnight thereby forming a radioactive complex with the specific IgE. The radioactivity is then compared to a standard. The Phadebas RAST reference system is composed of four dilutions of a pooled serum from patients highly allergic to birch antigen. Initially, reference A, the concentrated serum in this system, was assigned the arbitrary value of 50 Phadebas RAST units (PRU), and reference D, a 1-50th dilution of reference A, was assigned the arbitrary value of 1 PRU. The readability of the test was 1 to 100 PRU. More recently, the references have been improved extending readability to about 300 PRU (0.35-100 PRU). Serums with scores below reference D (originally 1 PRU and now 0.35 PRU) have been regarded as negative.
The RAST test is of particular utility in the management of pediatric allergic patients in whom end point skin test titrations are difficult to perform and which may be emotionally traumatic to both the child and his parents.
Despite the great value of the RAST test, it is deficient in that there is a very high degree of false negative results. For example, Deuschl and Johansson, Specific IgE Antibodies in Nasal Secretion from Patients with Allergic Rhinitis and with a Negative or Weakly Positive RAST on the Serum, Clinical Allergy, March 1977, reported on studies of 18 patients in whom a diagnosis of allergic rhinitis had been made by history and confirmed by intra-dermal skin testing and nasal provocation study. None of these patients were positive by the RAST test according to the cutoff value for positive result as determined by the Phadebas RAST curve. Many clinicians have had the disappointing experience of submitting known allergic serum for RAST testing and having the results returned to them as negative.
It is the object of this invention to provide a modified RAST test whose results can be used to determine a safe initial immunotherapy dosage and also an optimum dosage. It is also the object of this invention to provide a modified RAST test in which the number of false negatives is greatly reduced. This and other objects of the invention will become apparent to those skilled in the art from the following detailed description.